1. Field of the Invention
The present invention concerns engineered antibodies, with three or more functional antigen binding sites, and uses, such as therapeutic uses, for such engineered antibodies.
2. Description of Related Art
Structure of Naturally Occurring Antibodies
Naturally occurring antibodies (immunoglobulins) comprise two heavy chains linked together by disulfide bonds and two light chains, one light chain being linked to each of the heavy chains by disulfide bonds. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains (three or four constant domains, CH1, CH2, CH3 and CH4, depending on the antibody class). Each light chain has a variable domain (VL) at one end and a constant domain (CL) at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. See FIG. 1 herein. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains, see e.g. Chothia et al., J. Mol. Biol. 186:651-663 (1985); and Novotny and Haber, Proc. Natl. Acad. Sci. USA 82:4592-4596 (1985).
The constant domains are not involved directly in binding the antibody to an antigen, but are involved in various effector functions, such as participation of the antibody in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). The variable domains of each pair of light and heavy chains are involved directly in binding the antibody to the antigen. The variable domains of naturally occurring light and heavy chains have the same general structure; each comprising four framework regions (FRs), whose sequences are somewhat conserved, connected by three complementarity determining regions (CDRs) (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., (1991)). The four FRs largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen binding site.
FIGS. 2A-E herein depict the structures of the five major naturally occurring immunoglobulin isotypes. IgG, IgD and IgE immunoglobulins possess only two antigen binding sites. IgA and IgM, on the other hand, are capable of forming polymeric structures with higher valencies.
IgM is secreted by plasma cells as a pentamer in which five monomer units are held together by disulfide bonds linking their carboxyl-terminal (C:4/C:4) domains and C:3/C:3 domains. The five monomer subunits are arranged with their Fc regions in the center of the pentamer and the 10 antigen-binding sites on the periphery of the molecule. Each pentamer contains an additional Fc-linked polypeptide called the J (joining) chain, which is disulfide-bonded to the carboxyl-terminal cysteine residue of 2 of the 10: chains. The J chain appears to be required for polymerization of the monomers to form pentameric IgM; it is added just before secretion of the pentamer. An IgM molecule can bind 10 small hapten molecules; however, because of steric hindrance, only 5 molecules of larger antigens can be bound simultaneously. The increased valency of pentameric IgM increases its capacity to bind such multi-dimensional antigens as viral particles and red blood cells (RBCs).
IgA exists primarily as a monomer, although polymeric forms such as dimers, trimers, and even tetramers are sometimes seen. The IgA of external secretions consists of a dimer or tetramer, a J-chain polypeptide, and a polypeptide chain called secretory component.